Distinctive characteristics of granulation tissue in laparotomy wounds with underlying oncological processes

This study aimed to investigate the effects of malignant neoplasms on the morphological characteristics of laparotomy wound granulation tissue in the muscular-aponeurotic layer. The study involved a sample of 34 deceased individuals who had undergone abdominal organ surgery. Biopsy samples were taken from the muscular-aponeurotic layer of the anterior abdominal wall and subjected to histological examination, including staining with hematoxylin and eosin and methylene blue/Chromotrope 2B using N.Z. Slinchenko’s method. Descriptive methods and morphometry were used to evaluate pathomorphological changes. The results suggest that malignant neoplasms significantly impede and decelerate the maturation of laparotomy wound granulation tissue. Surgeries performed at the late stages of abdominal organ malignant neoplasms result in an uneven and slow maturation of the tissue, characterized by a higher prevalence of lymphoid cells, increased blood vessel volume, reduced optical density of stained collagen fibers, and pronounced chromotropophilia of collagen fibers. These distinct features should be considered to prevent postoperative eventration, a complication that is more likely to occur in this patient group. Clinicians should be aware of the possible consequences of malignant neoplasms on laparotomy wound granulation tissue, which may require additional measures to prevent postoperative complications in these patients.


INTRODUCTION
Postoperative eventration remains one of the most dangerous and threatening complications of abdominal surgery despite advancements in modern medicine. The mortality rate associated with postoperative eventration is estimated at 24%, and some authors suggest it to be as high as 65% [1][2][3]. General risk factors for postoperative eventration include old age, comorbidities, urgent surgery, relaparotomy, pneumonia, diabetes mellitus, cachexia, etc. [4][5][6][7].
Patients with oncological diseases of the abdominal organs are known to suffer from secondary immune deficiency, cachexia, anemia, etc., which is likely to influence the rate of regeneration and the risk of development of purulent-septic complications in the postoperative wound [8][9][10][11].
Understanding the morphology of granulation tissue in patients with malignant neoplasms of the abdominal organs may aid in the prevention of postoperative complications. Therefore, this study aimed to investigate the morphological features of the granulation tissue of the laparotomy wound in the muscular-aponeurotic layer after the removal of malignant neoplasms of the abdominal organs [12,13].

MATERIAL AND METHODS
To achieve the research objective, we conducted a post-mortem examination of 34 deceased individuals who underwent abdominal organ surgery. The main group included 16 patients with malignant neoplasms of the abdominal organs at stages ІІІ-IV, who died within the first 7 days of the early postoperative period. The comparison group included 18 patients with acute non-oncological surgical pathology of the abdominal organs, who also died within the first 7 days of the early postoperative period [14,15]. Table 1 presents the distribution of patients according to the surgery performed on the abdominal organs. In JOURNAL of MEDICINE and LIFE this study, there were 19 (55,9%) females and 15 (44,1%) males. The average age of the individuals was 61.3 ± 2.08 years, and the patient groups were representative with respect to gender and age. All patients received standard postoperative treatment according to established protocols for patients with urgent surgical pathology of the abdominal organs during their hospitalization.

Methods of morphological examination
The biopsy material obtained from the muscular-aponeurotic layer of the anterior abdominal wall for histological examination was fixed in a 10% neutral formalin solution. Paraffinic sections were prepared and stained with hematoxylin and eosin to visualize tissue structure. Additionally, N.Z. Slinchenko's method of histological specimen staining with methylene blue/ Chromotrope 2B was used to identify collagen fibers and fibrin. The morphological changes were analyzed using a descriptive method [16], and morphometry was performed using the computerized program ImageJ 1.48 v for computed microdensitometry to determine the optical density (OD) of collagen fibers. The specific volume of blood vessels in the granulation tissue (%) was determined using computed plane geometry, while the number of different types of granulation tissue cells was counted based on the surface area of the histological section (10000 µm²) [17].

Statistical analysis
Statistical analysis was performed using Microsoft Excel and PAST software package. To assess normality, the Shapiro-Wilk test was conducted, and the Mann-Whitney test was utilized to compare differences between groups. A p-value of ≤ 0.05, which is generally accepted in medical-biological studies, was used as the threshold for determining the reliability of the results [18].

RESULTS
The microscopic pictures of the specimens obtained from deceased individuals in both groups during the first day of the early postoperative period showed comparable features, such as a high concentration of fibrin, minimal lymphocyte presence, and areas with hemorrhages ( Figure 1). However, on the 2-3rd days, uneven maturation of the granulation tissue was observed in both groups, characterized by the presence of very immature foci (corresponding to 2-3 days -predominantly lymphoid cells) and moderately mature foci (corresponding to 5-7 days -predominantly fibroblasts) ( Figure 2).
Methylene blue/Chromotrope 2В staining revealed elongated collagen fibers and an accumulation of fibroblasts, lymphocytes, and a number of lymphoid cells and macrophages, which is specific for the immature granulation tissue ( Figure 3). The microscopic examination also revealed evenly immature granulation tissue with a significant number of lymphoid cells and macrophages, along with a limited presence of fibroblasts (as illustrated in Figure 4).
The formation of collagen fibers was not well-defined, and a considerable number of lymphoid cells were observed using the Methylene blue/Chromotrope 2B-stained microscopic slides according to N.Z. Slinchenko's method ( Figure 5).
During the 4-5th day of the early postoperative period, microscopic images in the main group displayed relatively more fibroblasts, fewer irregularly located lymphoid cells, and subtle muscle fibers ( Figure 6). The slow maturation of blood vessels and marked chromotropophilia of collagen fibers were also observed ( Figure 7). In the comparison group, there was a regular distribution of lymphoid cells and fibroblasts, indicating a more uniformly formed granulation tissue ( Figure 8).
During the later stages of the early postoperative period (6-7th days), the microscopic examination in the main group showed a less mature granulation tissue compared to the comparison group, as evidenced by the higher concentration of lymphoid cells and fibroblasts, and a clear chromotropophilia of collagen ( Figure 9-11).
A morphometric examination was performed to obtain an objective comparison of the granulation tissue maturation processes in the muscular-aponeurotic layer of the laparotomy wound [19,20]. The findings are presented in Table 2. The microscopic examination of the main group revealed a higher number of cells during all observation periods, including lymphoid cells, plasmatic cells, macrophages, fibroblasts, and single polymorphonuclear leukocytes. The number of cells was significantly greater during the later stage of observation (4-7th days) in both groups, compared to the early stage (1-3rd days).
The results in Table 3 indicate a significantly lower optical density of the collagen fibers stained with Methylene blue in the microscopic slides from the main group. The optical density of staining of collagen fibers was higher during later observation periods, although this difference was statistically significant only in the comparison group.
Further analysis of the study results, as presented in Table 4, revealed a significantly higher specific volume of the granulation tissue blood vessels in the microscopic slides. This increase was observed in both groups during the later observation periods.

DISCUSSION
Despite advances in modern medicine, postoperative eventration remains one of the most dangerous and serious complications of abdominal surgery. General risk factors associated with the development of postoperative eventration include advanced age, comorbidities, urgent surgery, relaparotomy, pneumonia, diabetes mellitus, and cachexia, among others [21,22]. Surgery for abdominal wall eventrations is not considered as urgent in adult         patients. The maturation of laparotomy wound granulation tissue is characterized by uneven and slow development, with a reliable prevalence of lymphoid-type cells, an increase in the specific volume of blood vessels, and a decrease in the optical density of stained collagen fibers. Thus, summing up the results of the study it should be noted that malignant neoplasms considerably inhibit and slow down the processes of the laparotomy granulation tissue maturation. This is characterized by a reliable increase in the number of cells (especially of the lymphoid type), as well as an increase in the specific volume of blood vessels and a decrease in the optical density of the stained collagen fibers. These findings suggest that the vascular-exudative inflammation reaction is not effectively completed in patients with malignant neoplasms. In addition, these processes are accompanied by uneven maturation of granulation tissue, expressed by the chromotropophilia of collagen fibers [23][24][25][26].
Moreover, uneven maturation of the granulation tissue and marked chromotropophilia of the collagen fibers are registered in the microscopic slides of physical bodies of the deceased individuals who suffered from malignant neoplasms of the abdominal organs. When staining sectioned slides with water blue/chromotrope 2B, the microscopic analysis revealed the presence of elongated collagen fibers and accumulation of fibroblasts, lymphocytes, and a significant number of lymphoid cells and macrophages. These characteristics are indicative of the presence of immature granulation tissue, which can impede the process of wound healing.
Slower maturation of the laparotomy wound granulation tissue is explained by larger surgical trauma caused by a complete removal of a malignant neoplasm with underlying pathological changes in the body resulting in their considerable deterioration [27][28][29][30].
This distinctive characteristic should be carefully taken into consideration in order to prevent the development of postoperative n -number of observations; p -against the indices of comparison; p1 -against the indices of the 1-4th days of observation.  JOURNAL of MEDICINE and LIFE eventration, which is significantly more likely to occur in this patient population.

CONCLUSIONS
Our study found that surgical intervention for malignant neoplasms of the abdominal organs at advanced stages is associated with a slower and uneven maturation of the laparotomy wound granulation tissue. This was demonstrated by an increased number of cells, mainly lymphoid, an increase in the specific volume of blood vessels, and a decrease in the optical density of stained collagen fibers. Furthermore, marked chromotropophilia of the collagen fibers was observed. These findings indicate a higher risk of postoperative eventration in patients with malignant neoplasms of the abdominal organs at advanced stages compared to those without oncological pathology in the same location. Our study highlights the importance of careful monitoring and management of wound healing in this patient population to reduce the risk of postoperative complications.